DE3051068C2 - - Google Patents

Description

The invention relates to the subject matter of the claim.

More recently, there is a group of connections that commonly referred to as prostaglandins (hereinafter abbreviated as PG or PGs (plural)) in the foreground reason of the different physiological functions in le kicking mammalian bodies. However, because some of the PGs have short half lives or are not stable Problems occurred using PGs as pharmaceuticals.

There is a method for controlling PGs that are in le Benden mammalian bodies are generated, for example by Administration of aspirin (see Nature, No. 239: 33-34, 1972). Aspirin, however, is an inhibitor of cyclooxygenase, who participated in an earlier stage of metabolism of PGs so Aspirin is a blocker for all PGs. This In other words, aspirin is a regulator of the generation of all PGs can be called and it no activity with regard to an increase in production exercise of a particular PG or PG, so that a natural limit on the activity of aspirin is given as a regulator of PGs. Furthermore aspirin exercises inso remote side reactions, as in mammals disorders of the Gastric and intestinal tracts occur, so that problems with a Long-term administration of aspirin is the result.

From DE-OS 29 14 493 is known, pharmaceutical agents, containing the p-aminobenzoic acid derivatives, for treatment hyperglycemia, hyperlipemia, hypertension and ent use inflammatory diseases.

From DE-OS 29 21 327 is the use of p-aminobenzoic acid acid-N-D-xyloside containing pharmaceutical preparations known for the indication ranges indicated above.

In DE-OS 29 21 328 the use of p-aminobenzoic acid acid N-L-rhamnoside and in DE-OS 29 14 005 the use of o-aminobenzoic acid derivatives containing agents for described the indications already mentioned.  

The prior art discussed above is a Note on the use of p-aminobenzoic acid derivatives for the prevention and treatment of thrombosis as well ischemic cardiac or cerebral diseases remove.

The present invention is based on the object To provide compounds that are preventive and healing effect on thrombosis, ischemic heart disease or cerebral diseases show and no unwanted Cause side effects.

This task is accomplished through the use of substances of claim 1.

The present invention enables the use of active ingredients which are able to intervene in the regulation of the prostaglandins in the organism. These active substances consist of one or more compounds of the general formula (I) in which R¹ represents a residual group which can be obtained by removing OH from the 1 (α) or 1 (β) position of arabinose, xylose, rhamnose, glucose, Galactose, mannose or fructose, R² represents a hydrogen atom, alkyl of 1 to 4 carbon atoms or a physiologically acceptable metal.

In the above-mentioned general formula (I), R¹ represents the residue of a monosaccharide, which monosaccharide may be in either the D-form or the L-form and either an α- anomer or β- anomer or a mixture of these anomers.

In this general formula (I), R² is a hydrogen atom, a physiologically compatible metal, like Na, K, 1-2 Ca, 1-2 Mg, 1-3 Al or alkyl, such as methyl, ethyl, Propyl or butyl.

Examples of the substances to be used according to the invention are shown in Table I.

The chemical synthesis of the invention used Compounds are described by Inoue et al. in J. Agr. Chem. Soc. Japan, Vol. 25, pp. 59-63 and pp. 291-293 (1951) and in Chemical Abstracts, Vol. 48, columns 2001d and 2001i (1954).

For example, for the production a mixture of 5 g of p-aminobenzoic acid, 5 to 6 g of a Monosaccharides, such as L-arabinose, D-xylose, L-rhamnose, D-glucose, D-galactose, D-mannose or D-fructose, and 0.1 up to 0,5 g ammonium chloride, magnesium chloride, formic acid, Acetic or hydrochloric acid in 40 to 90 ml a 95 to 100% pure methanol or ethanol under a reflux condenser for indexing a condensation he hitzt. After the reaction is complete, the reaction becomes mix at room temperature or in a cool place let stand whereupon the deposited crystals through Filter the reaction solution to be filtered off. The Crystals are washed with water, ethanol or ethyl ether wash and then from methanol, ethanol or an aqueous Solution of methanol or ethanol recrystallized.

To the hydrogen atom of the carboxyl group one on this Manned compound using a Base to replace, it is preferable to a known method apply. The compound, for example p-aminobenzoic acid N-pyranoside, is dissolved in an aqueous ethanolic Lö dissolved and an inorganic salt of the solution to Effect of substitution added.  

Some physical properties of the compounds will be in J. Agr. Chem. Soc. Japan, Volume 26, pages 329 to 331 (1952) and Chemical Abstracts, Vol. 48, Column 2003a (1954) and described in Table I. The used in the table by way of example according to the invention Substances are hereinafter referred to as the samples 1 to 11 designated.  

Table I

Physical properties of the active ingredients

The invention will become more apparent from the accompanying drawings explained. It shows:

Fig. 1 shows the effect of a substance according to claim 1 on the aggregation of platelets by arachidonic acid;

Fig. 2 shows the effect of a substance according to claim 1 on the aggregation of platelets by adenosine diphosphate (ADP);

Fig. 3 shows the effect of a substance according to claim 1 on the aggregation of platelets by collagen;

Fig. 4 shows the effect of a substance according to claim 1 on the aggregation of platelets by epinephrine;

Fig. 5 shows the effect of a substance according to claim 1 on the aggregation of platelets by ristocetin;

Fig. 6 shows the effect of a substance according to claim 1 on the aggregation of platelets by thrombin;

Fig. 7 shows the effect of a substance according to claim 1 on the content of cyclic adenosine monophosphate (cyclic AMP AM) in the platelets;

Fig. 8 shows the effect of a substance according to claim 1 on the production of malondialdehyde (MDA) in the platelets;

Fig. 9 blasts the action of a substance according to claim 1 on the production of prostaglandin I₂ (PGI₂) in 3T3 fibroblasts;

Fig. 10 shows the effect of a substance according to claim 1 on the content of prostaglandin F₂ α (PGF₂ α) ;

Fig. 11 shows the effect of a substance according to claim 1 on the content of prostaglandin E (PGE);

Fig. 12 shows the effect of a substance according to claim 1 for preventing Ver aggregation of ADP-induced platelets.

As the substances are closer as below is explained, an extremely low acute oral toxicity own and show no mutagenicity, the cellu larval and humoral immunity of the host animal are not flows and also the gastrointestinal bacterial flora in consequence of the lack of antibacterial activity is not ge disturbs. Such a substance can be administered orally during a lan be administered time span, without causing the risk a mutagenesis or allergy exists.

The physiological properties of the substances used according to the invention are summarized below, in the following order:
(1) Acute toxicity, (2) antimicrobial activity, (3) mutagenicity, (4) delayed intradermal reaction, (5) antibody-producing activity, and (6) gastric action.

(1) Acute oral toxicity

The acute oral toxicity of these substances is under the use of groups of ICR-JCL mice  seeks to provide orally representative examples of fiction according to substances to be used forced as an aqueous solution in distilled water or as an aqueous suspension also in distilled water at predetermined dosages be administered. After observing the toxicologi symptoms, if such symptoms occur at all during 6 days and recording the mortality until the 7th day after administration, the LD₅₀ (acute oral) according to the method of Litchfield-Wilcoxon. Then an autopsy of the dead and living mice is made Obtaining information.

The results are shown in Table II. Like from the Table II is the LD₅₀ (acute oral) reprä sentative examples of substances used according to the invention very large, which shows that these substances are extremely low Possess toxicity.

Table II

Acute oral toxicity of representative substances used according to the invention

In an autopsy of dead and living animals were no abnormal findings detected.

(2) antimicrobial activity

The substances to be used according to the invention are distilled Water in a row of a two-fold dilution system dissolved. These dilute solutions come with a Agar medium mixed in 9 times the volume and the Mi cast into a Petri dish. A heart infusion agarmedium is used for bacteria and Sabouraud agar medium used for mushrooms. After painting with the Vor culture are the inoculated plates at 37 ° C for 20 to 24 h in the case of bacteria and at 25 ° C for 3 to 7 days in the case of mushrooms incubated, whereupon the growth under is looking for. There are the following microorganisms Ermitt development of the antimicrobial activity of the invention used substances to be used:

Pseudomonas aeruginosa IAM 1514
Escherichia colia IFO 12734
Staphylococcus aureus 209 P
Bacillus subtilis IAM 1069
Saccharomyces cerevisiae IAM 4207
Candida albicans ATCC 752
Trichophyton mentagrophytes IFO 6124
Aspergillus niger IAM 3001

As results of the above-described tests, it is confirmed provided that none of the tested drugs growth Inhibition of all microorganisms at one concentration of 1 mg / ml.  

(3) Mutagenicity

In a first stage, the sub punching by rec-assay (i) and in a second stage by reversion assay (ii).

(i) A strain of Bacillus subtilis M45 which has a Recombination can not heal, and a wild Strain of Bacillus subtilis H 17, one of the recombina who can maintain the repair activity the, without their spreads intersect, at the starting point a B-2 agar culture plate inoculated (manufactured by Dissolve 10 g of meat extract, 10 g of polypeptone, 5 g of Na trium chloride and 15 g of agar in 1000 ml of distilled water with a pH of 7.0). Then a paper disc with a diameter of 8 mm, containing 0.04 ml of an aqueous Solution of the substance in sterilized Water absorbed on the surface of the agar plate placed in the way that they are the starting point of the above Covered bacteria culture strips covered. The inoculated B-2 agar plate is kept at 37 ° C overnight and the Length of the growth-inhibited region measured. kanamycin is used as a negative control and mitomycin as posi used a comparative comparative sample. The results of the rec assay are shown in Table III.

(ii) The strains of TA 98 and TA 100 (which are both histidine require) of Salmonella typhimurium become a reversal tion of the reversion assay used.

In 2 ml of a soft agar culture medium (the medium itself contains 6 g sodium chloride and 6 g agar in 1000 ml distilled Water) containing 0.1 volumes of 0.5 mM aqueous solution Biotin and 0.5 mM histidine have been added 0.1 ml of bacterial suspension and 0.1 ml of an aqueous Lö solution of a substance mixed in, whereupon Mix the mixture on the minimum agar medium  tet. After 2 days of incubation at 37 ° C, the number of counted in reverse colonies. Furylfuramide (AF-2) is described as used positive comparative sample. The results of the Re Version Assay are shown in Table IV.

As can be seen from Table III, the show Substances a weak mutagenicity only in one high concentration of 5000 micrograms / disc. How out Table IV, is with respect to the rate of Occurrence of mutations by the sub used in the invention No difference to the rate of the reference sample determine that no substance has been added, in a high concentration of 5000 micrograms / Plate. These findings show that the Substances with regard to mutagenicity are safe.  

Table III

Result of the rec assay

Table IV

Results of the reversion assay

(4) Delayed intracutaneous reaction

To the effects of the substances on the Cellular immunity became the foot paw response test using ICR-JCL mice as experimental animals and erythrocytes of sheep performed as antigen.

First, a mouse is injected by injecting 0.2 ml of a aqueous 10% suspension of sheep erythrocytes in a physiological saline solution in the tail vein subjected to a first sensitization, followed by 7 days after the first sensitization, 0.05 ml of an aqueous 40% suspension of erythrocytes of sheep in one physiological saline in the foot paw to Bewir kung a second sensitization are injected. The thickness of the foot paw is determined the next day. The Administration of the substances takes place in a dosage of 250 mg / kg / day once a day during consecutive 5 days from the day on, on which the first sensitization was performed.

The increase in the thickness of the footpaw of the mouse, to which egg ne substance has been administered shows no noticeable difference to the increase in the mice group to which no active substance has been administered.

(5) antibody-generating activity

To the effects of the substances on the To determine humoral immunity was the hemagglutinia carried out using ICR-JCL mice, sensitized with sheep erythrocytes.

A mouse was injected by injecting 0.2 ml of an aqueous 10% solution of erythrocytes of sheep in a physio logical saline in the tail vein sensitized. 7 days after the sensitization, a blood test for  the hemagglutination test to determine the antibodies taken from generating activity. The Substances were administered on five consecutive days ranges from the day of sensitization, and although intraperitoneally at a dosage of 250 mg / kg / day.

There was no noticeable difference in agglutination extent between the group to which the Substances have been administered, and the comparison group determined.

(6) effect of the substances on the stomach

5 g of the substances (each of samples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11) and aspirin are each because of beagles with a body weight of 9 to 12 kg in the form of an aqueous suspension in distilled water, adjusted to a pH of 3, administered. 90 minutes after administration, the dog's stomach is on a bleeding examined. It is found that only one bleeding in the Stomachs of dogs is to be given to aspirin has been reached while no bleeding in the stomachs of the Dogs can be found, to which one of the samples 1 to 11 has been administered.

Below are pharmaceutical peculiarities of he used according to the invention, in particular from the Examples emerge explained.

(1) Control of prostaglandins

These substances control prostaglandins, abbreviated as PG or PGs (plural) such as PGA, PGB, PGC, PGD, PGE, PGF, PGG, PGH, PGI, TXA and TXB as well as their metabolites. Further do not just control one of the PGs mentioned above, but also different types of PGs.  

The regulating function of the invention used Substances on the generation of PGs and their substance Alternatives are the result of the following facts:

(a) Suppression of aggregation of platelets

It is known that the aggregation of platelets main is reduced by PGG₂, PGH₂ and TXA₂. It became de found that these substances the Aggregation of the platelets to suppress (cf. Example 2).

(b) Increasing cyclic adenosine monophosphate

It is known that cyclic adenosine monophosphate (cycli AMP) in a close relationship to PGs, esp which stands for PGD, PGE and PGI. The sub used in the invention Punching shows a function in terms of increasing the content of cyclic AMP in platelets and cells in a culture (see Example 3).

(c) suppression of the production of malondialdehyde

It was found that these substances an inhibiting effect on the production of malondial dehydrate (MDA) in the platelets, using MDA as the substance A change product of PGs (see Example 4) is known.

(d) Accelerating the production of PGI₂

It was found that these substances accelerating the production of PGI₂ in cells, grown in vitro (see Example 5).

(e) investigations concerning the generation of some PGs

Experimental results concerning the metabolism of PGs in vitro using arachidonic acid as starting material have shown that the sub stances according to the invention the production of PGD₂, PGE₂, 6-keto-PGF _{1 α} and PGF _{2 α} (see Example 1) influence.

(f) action on the biosynthesis of PGE and PGF _{2 α}

In an in vitro culture experiment of cells, it was possible to show that the substances to be used according to the invention have an effect on the biosynthesis of PGE and PGF _{2- α} (compare FIGS. 10 and 11).

(g) Reinforcing effect on pigment transfer

In an attempt in the execution of an inventive Substance administered to tumors containing test animals has been the extent of the transfer of a Dye has been examined in the tumor tissue of the animal is a reinforcing effect of the transfer of the exogenous dye, resulting in a blood vessel erwei suggesting effect of the substances according to the invention.

In general, PGs in different organs of the Whole body found, from which their close relationship on the functions of the institutions. It is further known that the physiological and pharmacological radio These PGs cover an extremely wide range. This means that the PGs are mainly based on the extension and Constriction of blood vessels due to their pharmacological act on the circulatory system, with PGE and PGI act stronger in terms of expansion and in contrast TXA contracts the artery.  

Although that too strong progression of the effect of an aggre of the platelets atherosclerosis, a cerebral Infarction, a myocardial infarction and a cerebral apo Plexie causes, the PGs have a strong effect on the Slide out. This means that PGD, PGE and PGI have an anta gonistic function against the effect of aggregation exercise of platelets. TXA₂, PGG₂, PGH₂ and PGE₂ practice one inducing or accelerating effect on the aggregate tion. Therefore, by an appropriate attitude of the PGs a treatment and prevention of the above diseases are possible.

As already mentioned, cover the pharmacological Functions of PGs different areas, therefore, lets the fact that the substances PGs re Gulp, the expectation that PGs for prevention and for the treatment of the diseases described above are suitable.

On the other hand, from Example 1 show that the substances a Function regarding the change in the amount of both PGD as at PGE in cells, d. H. a function to the Steue exercise of PGs that are in the same cell from each other is different. Another example shows that the substances oppressive or be accelerating on the TXA and PGI act. this fact clearly shows that the substances are not change only a single prostaglandin, but also change different PGs at the same time.

One such substance containing many types of prostaglandins changed at the same time, has not been known so far. Such a property is specific to the inventions According to the substance to be used.

The formulation of the active ingredients for Her position of pharmaceutical agents according to the present Invention explained.  

The pharmaceutical active ingredients used according to the invention can be administered orally or parenterally, preferably orally. The invention according to the pharmaceutical active ingredients used can be in the form of powders, granules, Tablets, sugar-coated tablets, capsules, Suppositories, suspensions, solutions, emulsifiable con centraten, in the form of ampoules, injection solutions etc. be formulated. As a diluent every party comes substance, any liquid or semi-solid in question, For example, drawers, binders, Benet agents, decay-promoting agents, surface-active agents Agents, soaps, dispersants, buffering agents, Perfumes, preservatives, dissolution aids and solvents. In addition, you can choose one or more of these Use auxiliaries in combination or in mixture.

The pharmaceutical active ingredients used according to the invention can be Any known method can be formulated with the quantity of the active ingredient generally between 0.01 and 100% by weight of the total formulation fluctuates.  

The dose of a composition used according to the invention depends on the age, the person and the condition as well as the fact whether it is a human or an animal, generally the following cans adhered to: for humans is the oral dose 0.1 to 1000 mg / kg body weight / day and preferably 1 to 500 mg / kg / day and the parenteral dose 0.01 to 200 mg / kg / Day and preferably 0.1 to 100 mg / kg / day, divided into 1 to 4 parts, with 1 part administered at once.

Below are the formulations that make so like the physiological activities of the pharmaceutical Preparation according to the present invention with reference to explain in more detail.

Formulation Example 1 (part means part by weight)

The following components are mixed evenly, followed by the mixture to a powdered formulation with a medium size of less than 350 μ pulverized or is finely granulated.

(1) Sodium p-aminobenzoate-N-D-galactoside 10 parts (2) Heavy magnesium oxide 15 parts (3) galactose 75 parts

The formulation prepared in this way is described in the present form or used after encapsulation.

Formulation Example 2

The following components are mixed evenly, pul Verified and processed into a moist granules. Then followed by drying and screening of the grains with a Size from 177 to 1410 μ:

(1) Sodium p-aminobenzoate-N-D-xyloside 45 parts (2) strength 15 parts (3) galactose 16 parts (4) Crystalline cellulose 21 parts (5) polyvinyl alcohol 3 parts and (6) water used to prepare the wet granules 30 parts

Formulation Example 3

Instead of sodium p-aminobenzoate-N-D-xyloside in the case of Formulation Example 2 is sodium o-aminobenzoate-N-L- rhamnoside for the preparation of a granular formulation such as used in Formulation Example 2. To 96 parts of the This granular formulation prepared in this way becomes 4 Parts of calcium stearate were added, whereupon the mixture was added to Ta blanks with a diameter of 10 mm are compression-deformed becomes.

Formulation Example 4

To 90 parts of the prepared according to Formulation Example 2 Grainy formulation will be 10 parts of crystalline cellulose and 3 parts of calcium stearate are added, followed by these Mixture prepared into tablets with a diameter of 3 mm compression deformation. The way this way Tablets made with a mixed suspension made of syrup and gelatin and precipitated calcium carbonate to produce sugar-coated tablets tet.

Formulation Example 5

The following components are mixed well while heating, whereupon the mixture is filled into an ampoule and for In  for sterilization purposes:

(1) Sodium m-aminobenzoate-N-L-rhamnoside 0.6 parts (2) nonionic surfactant 2.4 parts and (3) aqueous physiological saline 97 parts

Example 1 Effect of a substance used according to the invention on the PGs metabolism sel of arachidonic acid, which has been incorporated into lymphocytes is.

After adjusting the lymphocytes from the spleen of a BALB / C mouse has been taken to a concentration of 1 x 10⁷ cells / ml become 2 μCi of ³H-arachidonic acid added and the mixture at a temperature of 37 ° C. incubated for 90 minutes. The incubated lymphocytes are washed three times with the culture medium. After a readjust the cells to a concentration of 1 × 10⁷ cells per ml, the culture in 4 is made from silicone dressed test tubes in an amount of 2 ml / tube filled. Two test tubes are used for comparison. In each of the remaining two test tubes are 500 .mu.g the substance, in this example the Sample No. 7, added to which the four test tubes at Be incubated at 37 ° C for 60 minutes. After the incubation who the test tubes at 0 ° C for 5 minutes at 1200 rpm zen trifugiert.

The cell pellets obtained in this way are in a Filled in vial containing 2 ml of culture medium. To the addition of 5 ml of petroleum ether shakes the vial and remove the ether layer. The remaining aqueous Layer is adjusted to a pH of 3.5 with an aqueous 0.5 n Hydrochloric acid solution adjusted.  

The aqueous acid solution is extracted three times with 5 ml of ether each time and the ether extract is dried to obtain a solid. The solid is esterified with a solution of diazomethane. The esterified reaction mixture is subjected to thin layer chromatography and developed with a mixed solvent of ethyl acetate, isooctane, acetic acid and water (90: 50: 20: 100 by volume). The identification of the spots occurring in this way on the chromatogram is _α and 6-keto-PGF _{1 α} leads Runaway using authentic samples from PGD₂, PGE₂, PGF _2. The silica gel layer of the separated chromatogram is scraped off and dissolved in a liquid for a liquid scintillation counter. From the change of the counts the change of the PGs is determined. The results are shown in Table V.

Table V

Changes in prostaglandins in cell culture in vitro

Similar results are obtained in cases where Samples 1 to 6 and 8 to 10 in an amount of 500 μg / ml be used. These results show that the respective Substances according to the present invention metabolism of PGs, even in an in vitro test, regu lose.

example 2 Effect of a substance to be used according to the invention on the aggregation of platelets

Since it has been found that the aggregation of Plätt Chen mainly by PGG₂, PGH₂ and TXA₂, respectively fall into a PGs series, the We kung a substance of the invention on the aggregation of platelets examined as follows:

As an anticoagulant, an aqueous citrate solution was used for the determination of erythrocyte sedimentation. To 1 part of the citrate solution was added 9 parts of freshly collected human blood. The mixture was centrifuged at 400 x G for 6 minutes and the supernatant layer used to prepare a human platelet rich plasma (PRP). The remainder was further centrifuged at 700 x G for 20 minutes and the supernatant fluid classified as platelet poor plasma (PPP). The change in permeability of PRP is measured in an Agrigometer (type PAP-3, manufactured by Biodata Co.) to determine the extent of aggregation of the platelets, the aggregation being effected by adding an agglutinating agent.

The used agglutinating agents consist of Ara chidonic acid (1.64 mmol), adenosine diphosphate (50 μmol), Collagen (0.26 mg / ml), epinephrine (0.11 mmol), ristocetin (2.0 mg / ml) and thrombin (0.5 units per ml). Each of the  Agent will give the PRP 2 minutes after the addition of one of the Substances (samples Nos. 1 to 8) added puts.

The results are shown in FIGS. 1 to 6. As can be seen from these figures, each substance tested exhibits an inhibitory effect on the aggregation of the platelets caused by each agglutinating agent. The above-mentioned phenomenon shows that each of the substances controls the generation of PGs, in particular the generation of PGG₂, PGH₂ and TXA₂.

example 3 Effect of a substance to be used according to the invention on the content on cyclic adenosine monophosphate (cyclic AMP)

Cyclic AMP is closely related to PGs, where it is also known as an intracellular messenger. The relationship is closer to PGD, PGE and PGI. Therefore, in the following Example of the influence of a substance according to the invention on cyclic AMP in the platelet cells in the following Way examined:

A PRP (a plate-rich plasma) is prepared according to the above-mentioned method and centrifuged for 20 minutes at 700 × G to obtain a supernatant liquid called PPP (platelet-poor plasma). The precipitate is redistributed in 1/3 times the volume of PPP to produce a triple concentrated PRP.

To the thus prepared concentrated PRP, an aqueous solution of the substance according to the invention in a predetermined concentration (in this case, the samples 4, 7 and 8) and an aqueous physiological saline solution is added, after which the mixture is incubated at room temperature for 5 minutes , It is then boiled in the order given, homogenized and centri fuged, with 50 ul of the supernatant liquid for the measurement of cyclic AMP are used. The measurement is performed according to the method of Gilman. As a positive comparison sample PGE₁ is used. The results are shown in FIG. 7. As is apparent from Fig. 7, an increase in the content of intracellular cyclic cyclic AMP is found in the present case, it can be seen from this fact that the inven tion used according to the compound affects the metabolism of PGs.

example 4 Effect of a substance to be used according to the invention on the content of malondialdehyde (MDA) of platelets

PRP, which has been collected from human blood, is centrifuged and washed to obtain "washed platelets". After adding one of the inventive substances (Sample Nos. 4, 7 and 8) to the washed platelets, Caionophore A-23187 is added and the mixture is incubated at 37 ° C for 5 minutes. Then, thiobarbituric acid is added to the incubated mixture as a color developer, whereupon the mixture is extracted with a solvent mixture of methanol and butanol. The absorption at 535 nm is carried out colorimetrically for the determination of the amount of permondialdehyde (MDA). The results are shown in FIG. 8. This compound suppresses the generation of MDA. From this fact, the participation of the substances in the metabolism of PGs is also likely.

example 5 Influence of the substance used in the invention on the PGI₂ productivity of 3T3 fibroblasts

The influence of a substance according to the invention on the PGI₂- Productivity of mouse 3T3 fibroblasts is reduced examined. For comparison, a culture is used in which Arachidonic acid, the precursor of PGs, a culture medium of mouse 3T3 fibroblasts. The Mi is 5 minutes at 37 ° C to produce PGI₂ incubated.

On the other hand, 30 mmol each of the Substances (Samples 1, 3, 4 and 7 to 11) 4 ml of a Culture medium of 3T3 cells was added, whereupon the Mi incubated for 2 minutes at 37 ° C. After the addition from arachidonic acid to the incubated culture becomes this again as in the case of the comparative experiment for production incubated by PGI₂. The just mentioned culture is called "treated group".

After recovery of the respective supernatants The cultures will be the reference sample and the be acted group regarding their productivities of PGI₂ compared, with the suppressive effect on the aggre gation of the platelets is used as an indicator, the induced by the addition of 2.43 mmol arachidonic acid becomes.

The results are shown in FIG. 9. As shown in Fig. 9, although suppression of platelet aggregation is detected to some extent in the case of the control sample 5 minutes after the addition of arachidonic acid as a precursor of PGs, marked suppression is found in the case of the treated group, resulting in increased productivity of PGI₂ is manifested by the addition of the substance.

example 6 This is another example showing the effect of a used according to the invention on the PGI₂- Productivity of 3T3 fibroblasts explained

After excision of the neck aorta of an SD rat under Anesthesia is the aorta with Krebs-Linger bicarbonate buffer washed and cut into thin rings. The Cut pieces are in the above-mentioned Pufferlö in an amount of 1 mg of wet cuts kept per 0.5 ml for the production of PGI₂.

In 500 microlitres of Mi thus prepared mixture 50 microliters of an aqueous physiological saline solution containing 2 wt .-% of the substance (each of sample Nos. 4, 7, 8) in dissolved form added. Since the generation of PGI₂ generally holds its maximum after standing for 10 minutes, it keeps a supernatant liquid (2.5 μl) of the mixture made 10 minutes after the addition, whereupon the amount of PGI₂ in the supernatant liquid is suppressed by the suppression effect Aggregation of platelets as an index and using ADP is determined as an agglutinating agent, wherein an aqueous physiological solution is used as a comparative sample. The results are shown in FIG. 9. The addition of the substance increases the production of PGI₂ through the cut pieces of the Halsaorta.

There have already been various studies with regard to performed on the cause of atherosclerosis, where the agglutination of aggregated lumps on the wall the vessels are highlighted as a special cause. This In other words, preventing the Agglu the platelets on the wall of the vessels the treat is the choice of choice. It is known that PGI₂ has a strong in  hibierende effect against the agglutination of platelets owns. From the gained knowledge goes German Lich that the substances, which increase the producibility of PGI₂ on the aorta wall, an effect on the treatment and prevention of Exercise arteriosclerosis.

example 7 Improvement of erythrocyte deformability

3 hours after oral administration, each of those given in Table VI mentioned compounds in a dose of 300 mg / kg 5 Wistar rats of each group were given blood samples taken from the rats and these tenfold Volume of an aqueous physiological saline solution mixed.

After the mixture thus prepared was mechanically hemolyzed by stirring in a mixer, this hemolyzed mixture was centrifuged and the amount of hemoglobin (A ₁) in the supernatant liquid was measured colorimetrically. Independently, the blood samples were mixed with ten times the volume of distilled water in place of the aqueous saline solution, and the amount of hemoglobin (B ₁) in the supernatant liquid obtained by centrifugation of the stirred mixture was measured colorimetrically. The hemolysis rate (H _i ) was calculated according to the following formula:

The same measures were carried out for control purposes except that an aqueous physiological saline solution was applied instead of the sodium salt of the compounds according to the invention and the hemolysis rate (H _c ) was obtained from the blood of the controls.

The relative hemolysis rate of each of those shown in Table VI Compounds were calculated by the following formula and shown in Table VI:

As can be seen from Table VI, assign the groups of rats containing a sodium salt of Compounds had been administered, a lower Hemolysis rate as the control group, d. H. the Deformability of erythrocytes of rats from the According to the invention treated group is greater than that of the control group as shown in Table VI.

These results show an improvement in deformity erythrocytes after administration of each of those given in Table VI In particular, sodium-p- aminobenzoate-N-D-mannoside shows a strong effect.

An improvement in erythrocyte deformability leads to improve the blood circulation in the organism and accordingly, the administration of the sodium salts is the present compound for the treatment of patients with Cerebral ischemia suitable because the blood circulation at a patient with cerebral ischemia Minimum is reduced.

test substances Relative hemolysis rate Sodium-p-aminobenzoate-N-L-arabinoside 88 Sodium-o-aminobenzoate-N-L-arabinoside 90 Sodium-p-aminobenzoate-N-L-rhamnoside 70 Sodium-o-aminobenzoate-N-L-rhamnoside 70 Sodium-p-aminobenzoate-N-D-galactoside 79 Sodium o-aminobenzoate-N-D-galactoside 83 Sodium-p-aminobenzoate-N-D-xyloside 71 Sodium o-aminobenzoate-N-D-xyloside 77 Sodium-p-aminobenzoate-N-D-glucoside 80 Sodium o-aminobenzoate-N-D-glucoside 80 Sodium-p-aminobenzoate-N-D-mannoside 56 Sodium m-aminobenzoate-N-D-mannoside 79 control 100

example 8 Promoting effect on the coronary blood flow volume in the coronary sinus

After inserting a Morawity cannula into the coronary sinus Each of three beagle dogs was in the right atrium the coronary blood flow volume with an electromagnetic Flowmeter before and after administration of each of the in Table VII shown dissolved in a physiological saline solution Compounds measured at a dose of 100 mg / kg. The by subtracting the blood flow volume before administration of after the dose, divided by the blood flow volume the gift received, d. H. the slew rate the blood flow volume is shown in Table VII.

Table VII shows that each of the tested compounds exhibits coronary blood flow volume increasing activity, particularly effective are the compounds sodium p-aminobenzoate-ND-mannoside, sodium p-aminobenzoate-N-rhamnoside and sodium-aminobenzoate-ND-xyloside ,

Table VII - Contributing effect on coronary blood flow volume test substances Improvement (%) Sodium-p-aminobenzoate-N-L-arabinoside 22.7 Sodium-o-aminobenzoate-N-L-arabinoside 10.8 Sodium-p-aminobenzoate-N-L-rhamnoside 49.4 Sodium-o-aminobenzoate-N-L-rhamnoside 9.7 Sodium-p-aminobenzoate-N-D-galactoside 27.5 Sodium o-aminobenzoate-N-D-galactoside 26.0 Sodium-p-aminobenzoate-N-D-xyloside 38.3 Sodium o-aminobenzoate-N-D-xyloside 10.0 Sodium-p-aminobenzoate-N-D-glucoside 14.9 Sodium o-aminobenzoate-N-D-glucoside 20.1 Sodium-p-aminobenzoate-N-D-mannoside 50.2 Sodium m-aminobenzoate-N-D-mannoside 5.4

example 9 Inhibiting effect on the agglutination of platelets

Platelet-rich plasma was obtained from a fresh sample of human blood. After adding an aqueous physiological saline solution or a 50 mM solution of each of the compounds listed in Table VIII in an aqueous saline solution and each of three agglutination inhibitors for two minutes, the extent of platelet agglutination in the thus treated mixture was determined by of an agglomerometer. The extent of platelet agglutination inhibition ( _I.sub.i ) of each compound tested was calculated by dividing the extent of platelet agglutination prevented by the extent of platelet agglutination upon addition of physiological saline and multiplying the value obtained by 100. The results are set forth in Table VIII.

From Table VIII it can be seen that the Ver compounds have an inhibitory effect on platelet agglutination in the presence of each agglutinin, especially sodium p- aminobenzoate-N-D-mannoside shows a strong effect.  

TABLE VIII Inhibitory effect on platelet agglutination (inhibition rate,%)

example 10 Effect of preventing death by thrombotic embolism

After three hours of oral administration of 300 mg / kg each the compounds shown in Table IX to a mouse, Adenosine phosphate or mouse collagen was given intravenously injected and the mortality of the mice so treated certainly. As a control, a mouse was used which distilled water was administered orally. The results are given in Table IX. From Table IX It can be seen that each of the tested compounds the Mortality, in particular sodium p-aminobenzoate N-D-mannoside has a good effect.  

Table IX - Prevention of death from thrombotic embolism

Claims (1)

Use of a compound of the general formula wherein R¹ represents a residual group formed by removing OH in the 1 (α) or 1 (β) position of arabinose, xylose, rhamnose, glucose, galactose, mannose and fructose, and R² represents a hydrogen atom, a Alkyl group having 1 to 4 carbon atoms or a pharmaceutically acceptable metal, for a preventive and curative treatment of cerebral or myocardial infarction.